FIGURE 2.

Recombinant NRP-1 proteins stimulate tube formation in HUVEC and activate VEGFR-2 signaling. A and B, influence of addition of VEGF-A₁₆₅ (10 ng/ml), Fc and Fc rNRP-1 (5 nm), and shNRP-1 (5 nm and 10 nm) on HDMEC tube formation on collagen substratum (white bar indicates 100 μm). The tubes were photographed and analyzed as described under “Experimental Procedures.” Significant differences between the tested agonists and the control were determined by Student's t test. Single asterisk (*) indicates that the p value is <0.001 for comparison to control, double asterisk (**) indicates that the p value is <0.001 for comparison to Fc. C, Western blot for phosphorylated VEGFR-2 and RCAN-1.4 of cells from a parallel experiment conducted on a layer of collagen. Samples were collected for analysis 10 min and 3 h after addition of VEGF-A₁₆₅ (10 ng/ml), Fc, shNRP-1, and Fc rNRP-1 (10 nm). The band intensities were analyzed as described under “Experimental Procedures.”